When reactive substances such as protein, peptide and amino acid having an amino group(s) coexist with a reducing sugar such as aldose having an aldehyde group(s), they combine nonenzymatically and irreversibly through the amino and aldehyde groups, which is followed by amadori rearrangement to form an amadori compound. Examples of materials containing an amadori compound include food products such as soy sauce and body fluids such as blood. The production rate of an amadori compound being a function of concentration of reactants, contacting period, temperature and the like, various useful information about a sample containing such a reactive substance(s) can be derived from the amount of amadori compounds.
For instance, fructosylamines which are amadori compounds wherein glucose is bound to amino acid residue are formed in a living body. The so produced glycated derivatives of hemoglobin, albumin and proteins in blood are called glycohemoglobin, glycoalbumin and fructosamine, respectively. As the concentration of these glycated derivatives in blood reflects an average of blood sugar levels over a particular period of time, it can be used as a significant index for diagnosis and control of conditions of diabetes. Therefore, the establishment of a method of measuring an amadori compound in blood is clinically useful.
Further, a state of preservation and period after production of a food product can be estimated on the basis of the amount of amadori compounds in the food product. Accordingly, the method of measuring an amadori compound can also contribute to the quality control of a food product.
Thus, an assay of amadori compounds should be useful in wide range of fields involving medicine and food products.
There has been proposed an assay of amadori compounds which comprise reacting an oxidoreductase with a sample suspected to contain amadori compounds and determining oxygen consumption or hydrogen peroxide generation as an index of the amount of amadori compounds.
The decomposition of amadori compounds catalyzed by an oxidoreductase can be represented by the following reaction scheme: EQU R.sup.1 --CO--CH.sub.2 --NH--R.sup.2 +O.sub.2 +H.sub.2 O.fwdarw.R.sup.1 --CO--CHO+R.sup.2 --NH.sub.2 +H.sub.2 O.sub.2
wherein R.sup.1 is an aldose residue and R.sup.2 is an amino acid, protein or peptide residue.
The enzymatic assay of amadori compounds and enzymes usable therefor are well known in the art from literatures, such as Japanese Patent Publication (KOKOKU) No. 5-3399, Japanese Patent Publication (KOKAI) Nos. 61-268178, 2-195900, 3-155780 and 2-195899.
However, the existing assays and the enzymes are not necessarily useful for a particular purpose. It is needed to select the most suitable enzyme for individual purpose so as to perform the determination of an amadori compound correctly and efficiently. For example, the glycoalbumin level reflects the mean glycoprotein value of for past 1 to 2 weeks and it is desirable to use an enzyme with higher substrate specificity to fructosyl valine than fructosyl lysine in glycated protein in blood for the diagnosis of diabetes. However, such an enzyme has not been provided so far. The above-mentioned Japanese Patent Publication (KOKAI) 3-155780 discloses an enzyme from Aspergillus having molecular weight of about 80,000 to 83,000, but the enzyme is less active on fructosyl lysine compared to fructosyl valine.
On the other hand, an enzyme active on both of fructosyl valine and fructosyl lysine is preferred for the determination of glycated hemoglobin.
The present inventors have intensively studied for purposes of providing an enzyme useful for establishing the purposes above, and have purified a fructosyl amino acid oxidase (FAOD) from Fusarium and disclosed the usefulness thereof (Japanese Patent Publication (KOKAI) 8-154672 corresponding to EP-A-709457); Japanese Patent Publication (KOKAI) 7-289253 corresponding to EP-A-678576), and from Aspergillus (PCT/JP96/03515). The inventors have found that these FAODs contain an enzyme which is more specific to fructosyl lysine than fructosyl valine, for example, the one produced by Aspergillus terreus GP1 (FERM BP-5684), and named the enzyme of this kind "FAOD-L". As the FAOD-L was expected to be useful for diagnosis of diabetes, the present inventors have continued research on it.
However, it requires a plenty of labor and time to grow a microorganism such as a strain of Aspergillus in a medium and purify an enzyme from the culture, and is inefficient. In addition, an enzyme isolated from the culture is probably accompanied with contaminants such as proteins originated from the strain of Aspergillus, which can contain a substance capable of affecting the FAOD activity reversely, and would reduce the reliability of assay.
A purified FAOD originated from a microorganism can be obtained efficiently by means of DNA recombinant technology which comprises cloning a DNA encoding an FAOD, constructing an appropriate expression vector containing the DNA, transforming an appropriate host cells by the expression vector, and culturing the transformant in an appropriate medium. However, DNA encoding an FAOD originated from Fusarium or Aspergillus has not been cloned prior to the present invention. Accordingly, it was necessary to isolate DNA encoding an intended FAOD from a microorganism.